Molecular characterization of selected enterococcus strains (previously streptococcus) using genotyping techniques.
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Date
2007
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Abstract
The genus Enterococcus comprises of a group of commensal organisms of the human
gut which has been associated with cases of endocarditis and urinary tract infections.
In the present study, 12 Enterococcus isolates were obtained from clinical specimens
and characterized using genotyping techniques that have become an integral part of
clinical research. There were three different genotyping methods used to identify the
enterococci to species level and to determine the level of genetic diversity among the
selected strains. These techniques were, randomly amplified polymorphic DNA-PCR
(RAPD-PCR), 16S rDNA ribotyping analysis and pulse field gel electrophoresis
(PFGE) respectively. The minimum inhibitory concentration (MIC) to penicillin and
vancomycin were also determined using a disc diffusion assay and a microtitre plate
dilution assay. All twelve strains were found to be vancomycin resistant enterococci
(VRE) at a MIC value greater than 100μg/ml. Penicillin growth inhibition based on
MIC values were categorized into three groups, susceptible (< 0.25 μg/ml),
intermediate (≤ 3μg/ml) and resistant (≥ 4μg/ml) respectively. RAPD-PCR was
performed using four random primers. Primers yielding the highest discriminative
power were used for phylogenetic analysis. The phylogenetic analysis indicated that
all 12 strains yielded clonal dissemination, therefore a low genetic diversity between
them. The 16S rDNA of all strains were used to identify the enterococci at species
level. The rDNA were sequenced and analysed using the NCBI BLAST algorithm and
found to belong to three species of Enterococcus. These were E.faecalis, E.faecium
and E.durans. PFGE analysis was performed by restriction of all 12 strain’s genomic
DNA with the restriction enzyme SmaI. The PFGE patterns were divided into two
groups with low genetic diversity. Compared with the RAPD PCR patterns PFGE
gives a higher discriminatory power as a higher dissimilarity between the strains was
observed. Similar penicillin MICs for each of the strains in the three categories are
grouped together in the phylogenetic trees for both PFGE and RAPD-PCR. RAPDPCR
is a sensitive, faster, specific and cost effective technique, PFGE analysis has
given a higher discriminatory power, higher reproducibility of the results and the
polymorphism seen in the patterns suggest that PFGE has a potential of being an
essential tool in clinical diagnostics.
Description
Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2007.
Keywords
Enterococcus--Molecular genetics--Technique., Streptococcus., Polymerase chain reaction., Gel electrophoresis., Diagnosis, Laboratory., Clinical medicine--Research., Theses--Genetics.